HPLC-FTIR is a strong conceptual synergy in the analytical approach of complex non-volatile or thermally labile mixtures. The main aim of this hyphenation is to eliminate the drawback of the conventional offline analysis, which involves fractionated fractions being pooled; the solvent is then dried, and the residue is measured using IR. This is a time-consuming offline process, which may result in loss or degradation of samples or may add artifacts.
Principles and Interface Challenges
The basic concept of HPLC-FTIR is the following: the outflow of an HPLC column is repeated in an FTIR spectrometer to receive analysis on the fly. But the major challenge with the implementation is associated with the extreme IR absorption of the mobile phase, which is the liquid phase. The most frequently used HPLC solvents, like water, acetonitrile, and methanol, have very intense and wide spectral regions in the IR spectra, which may saturate the important spectral regions of the analyte. This is the greatest problem in HPLC-FTIR: this is the solvent interference problem. To overcome this, special interfaces have been created. The two most widespread methods are the flow cell interface and the solvent elimination interface.
Interface Technologies: Flow Cell vs. Solvent Elimination
The interface selection determines the functions and the restrictions of an HPLC-FTIR system. The flow cell interface works by directing the whole HPLC effluent in a miniature, IR-transparent flow cell that is embedded into the light beam of the FTIR. Peaks of the spectra are eluted, and continuous collections of the spectra are achieved. Although this enables data to be attained in real time, its second serious constraint is that it necessitates IR-transparent phases of the mobile that severely limit the adaptability as well as the maximization capability of the HPLC technique. It can only be usually applied in chromatography at normal phase using halogenated or hydrocarbon solvents. The Solvent Elimination Interface is a more sophisticated and flexible method in contrast. This configuration involves deposition of the HPLC effluent onto a surface, and the volatile mobile phase is evaporated.
Applications
Although it has its issues, HPLC-FTIR has useful niches in the areas where the molecular structure, and especially the identity of functional groups, is primary, and other methods, such as LC-MS, are potentially ambiguous. It is applied in the analysis of polymers to define copolymers and additives to determine the chemical character of various eluting fractions. It is also applicable in the pharmaceutical industry to detect isomeric impurities or degradation products of the same mass with different functional groups. It may be used in the characterization of complex mixtures of surfactants or hydrocarbons in environmental analysis.
Comparison with Other Hyphenated Techniques
Liquid Chromatography-Mass Spectrometry (LC-MS), being an omnipresent method, takes up a less dominant position compared to HPLC-FTIR. LC-MS is very good at giving molecular weight and fragment patterns with excellent sensitivity and operates effectively with a broad variety of aqueous mobile phases. HPLC-FTIR is a direct solution to this shortcoming since it enables the offering of direct data regarding the structure of the molecule, bonding, and functional groups.
Advantages
The major benefit of HPLC-FTIR is that it offers direct and informative structural fingerprints of chromatographically separated components, which is priceless in the process of identifying unknowns and differentiating among isomers. When a flow cell is used, the method is non-destructive, and the solvent removal method generates a permanent and re-analyzable record of the separation.
Limitations
The interface technology, especially in solvent removal, is complicated and costly, and must be optimized well to facilitate efficient solvent removal and quantitative deposition. The problem of phase compatibility in a mobile phase is another major limitation that is mostly detrimental to reversed-phase chromatography, particularly when using the flow cell method.
Conclusion
HPLC-FTIR is an extremely specialized hyphenated method that combines fine resolution separation with in-depth molecular spectroscopy. Although its development and acceptance have been stunted by the daunting task of solvent interference, more sophisticated interfaces of solvent elimination have rendered it a powerful and practical tool for analyzing selected analytical issues. It has the special advantage of being able to give unambiguous functional group and structural data, which supplements data obtained by mass spectrometry.
